Microduplication and triplication of 22q11.2: a highly variable syndrome

Abstract

22q11.2 microduplications of a 3-Mb region surrounded by low-copy repeats should be, theoretically, as frequent as the deletions of this region; however, few microduplications have been reported. We show that the phenotype of these patients with microduplications is extremely diverse, ranging from normal to behavioral abnormalities to multiple defects, only some of which are reminiscent of the 22q11.2 deletion syndrome. This diversity will make ascertainment difficult and will necessitate a rapid-screening method. We demonstrate the utility of four different screening methods. Although all the screening techniques give unique information, the efficiency of real-time polymerase chain reaction allowed the discovery of two 22q11.2 microduplications in a series of 275 females who tested negative for fragile X syndrome, thus widening the phenotypic diversity. Ascertainment of the fragile X-negative cohort was twice that of the cohort screened for the 22q11.2 deletion. We also report the first patient with a 22q11.2 triplication and show that this patient's mother carries a 22q11.2 microduplication. We strongly recommend that other family members of patients with 22q11.2 microduplications also be tested, since we found several phenotypically normal parents who were carriers of the chromosomal abnormality.

Figure 1

Duplications and deletions on chromosome 22q11.2. Arrows indicate the size of the various deletions and duplications. Triangles below the line representing the chromosome show the location of the LCR22s. Probes located above the line were used in the various FISH and dosage techniques.

Figure 2

Photographs of patients with increased dosage of 22q11.2. A, Patient 1. B, Patient 4. C, Hands of patient 4 and her mother (patient 4M). The unlabeled hand is an unaffected sister of patient 4.

Figure 3

FISH analyses. A, Patient 1 test probe TUPLE1 (red) shows three copies for an interphase cell; control probe ARSA (green) shows two copies. B, Patient 4 test probes 361L10 and 901P22 (green and red, respectively) show four copies for this cell. C, Patient 5 test probe TUPLE1 (red) shows three copies; control probe ARSA (green) shows two copies. D, Metaphase spread from patient 5 shows the duplication of the TUPLE1 probe.

Figure 4

Microsatellite analysis of probe D22S1709, showing four different alleles in patient 4, with sizes (in bp) marked below the peaks. Unmarked peaks are due to stutter bands, an artifact of PCR. The method has been described elsewhere (Edelmann et al. 1999a).

Figure 5

Real-time PCR analysis of probes COMT and HIRA (also known as “TUPLE1”). Copy number was calculated by multiplying the ΔΔCt ratio by 2. A two-copy control was run for each set of experiments, but only the most variable two-copy control is shown in each graph. Error bars represent 1 SD above and below the mean. Each reaction mixture consisted of 200 nmol/L of each primer, 50 nmol/L of TaqMan probe, 40 ng of genomic DNA, 0.3 μl of PDAR (Applied Biosystems), and 7.5 μl of 2× TaqMan mix (Applied Biosystems), in a total volume of 15 μl. Thermal cycling conditions included a presoak for 2 min at 50°C and for 10 min at 95°C. Samples were amplified for 40 cycles at 95°C for 15 s and at 60°C for 1 min.

Figure 6

MAPH analysis of the 22q11.2 region for patients 1, 2, 3, and 4. Probes are shown below the four graphs and are listed in order along the chromosome (centromere at left), with the CES region and the 22q11.2 deletion syndrome region indicated. Normalized mean peak ratios (error bars represent 1 SD above and below the mean) are graphed for all probes for each patient (unblackened triangles connected by a line) and control (two-copy control sample represented by blackened diamonds; three-copy control sample represented by squares). The four-copy control sample (circles) has four copies of the CES region and the 22q11.2 deletion syndrome region; however, distal to the 22q11.2 deletion syndrome region, the copy number is 2. Probes and probe mixes, membrane preparation, and hybridization were performed in accordance with the study by Armour et al. (2000) and the Multiplex Amplifiable Probe Hybridization (MAPH) Web site, with the following changes. Genomic DNA was blotted onto a Hybond N membrane (Amersham Biosciences). After hybridization, the membranes were washed six times for 10 min each at 65°C in 1× SSC/1% SDS solution and six times for 10 min each at 65°C in 0.1× SSC/0.1% SDS solution. Membranes were each placed in a 0.2-ml PCR tube with 50 μl of 1× AB gene buffer IV and were heated to 95°C to release the probes; 3.75 μl of this solution was used as a template for a fluorescent PCR containing 1× AB gene buffer IV, 2 mM MgCl₂, 0.4 mM dNTPs, 2 μM PZA-FAM, 1 μM PZB, and 2.5 U Taq polymerase (Invitrogen). Finally, 1 μl of the PCR was mixed with 1 μl of formamide loading dye, and 1.2 μl of the mixture was run on a polyacrylamide gel for 3.5 h by use of the ABI Prism 377 DNA sequencer.